1. Field of the Invention
This invention relates to a method of binding a biologically active material, e.g. a protein to a carrier containing hydroxyl groups, wherein an isocyanate compound is bound to the carrier and whereafter the said biologically active material is bound to the so bound isocyanate compound.
2. Background of the Invention
For several inherent reasons it has been necessary to use special methods for binding enzymes to a carrier. One reason is that enzymes are sensitive substances and easily deactivated, requiring the use of mild reaction conditions for the binding. Hydroxyl groups on polymeric materials are very inert and must be attacked by rather aggressive reagents to be used as anchoring groups. Of course, only very strongly reactive groups can therefore take part in the binding reaction. Enzymes to be bound are usually handled in the form of buffered dilute aqueous solutions. This entails the risk that such anchoring groups may be consumed by side-reactions with the large excess of water present.
According to the prior art techniques, some compensation had to be found for such an inherently low reaction efficiency. Particularly, in connection with carriers containing hydroxyl groups, for example polysaccharides, this has been achieved by means of some form of activation of the hydroxyl groups. The most common form for activating has been by means of cyanogen bromide, as described in Axen, Ernback: European J. Biochem. 1971, Vol. 18, p. 351.
Although such activating has made it possible to solve the above-mentioned problem, the Axen-type method has entailed other serious disadvantages and risks. As is apparent from scheme 2 in the Axen et al. reference, the bonds are not very stable. The ligands can be split from the carrier, either hydrolytically or especially ammonolytically.
Moreover, contaminating cyanide ions from the cyanogen bromide can affect the activity of some enzymes by blocking central metal atoms. See, for example, K. Pommerening et al., J. Pol. Sci.: Polymer Symp., Vol. 66, pp. 185-188 (1979).
Furthermore, cyanogen halogenides are highly toxic and comparable with hydrocyanic acid in this respect. The use of the strongly toxic and highly volatile (b.p. 61.degree. C.) cyanogen bromide according to the prior art technique therefore requires special measures for safety, especially when being used in industrial quantities.
For steric reasons, some enzymes also require an anchorage to the carrier via a long-chain linkage ("spacer"). In order to provide such an anchorage according to the known BrCN-method, it has therefore been necessary to employ a separate second reaction step, i.e., first to bind a spacer and finally to bind the biologically active material to this spacer.
Finally, the BrCN-method, when used in connection with cellulose-type carriers, has required that the cellulose has firstly been exposed to an alkaline treatment, i.e., mercerizing. See, for example, J. A. Jackson et al., J. Pharm.+Exp. Therapeutics, Vol. 209, 2 (1979), p. 271.
Another technique for binding proteins to a carrier is disclosed in Biebricher et al. U.S. Pat. No. 4,177,038. In the Biebricher et al. method, a vehicle containing free --NH.sub.2 or --OH groups is reacted under anhydrous conditions with an excess of organic diisocyanate in the presence of a strong basic catalyst to produce vehicle substance which can be reacted with biologically active matter, e.g., protein. The only specific catalyst mentioned is sodium imidazolide. Biebricher et al. also suggest the use of a "spacer" compound.